Antimicrobial Face Masks and Mask Covers with a Salt-Coated Stacked Spunbond Polypropylene Fabric: Effective Inactivation of Resilient Pathogens and Prevention of Contact Transmission.

In response to the ongoing threat posed by respiratory diseases, ensuring effective transmission protection is crucial for public health. To address the drawbacks of single-use face masks/respirators, which can be a potential source of contact-based transmission, we have designed an antimicrobial face mask and mask covering utilizing a stack of salt-coated spunbond (SB) fabric. This fabric acts as an outer layer for the face mask and as a covering over a conventional mask, respectively. We evaluated the universal antimicrobial performance of the salt-coated three-stacked SB fabric against enveloped/nonenveloped viruses and spore-forming/nonspore-forming bacteria. The distinctive pathogen inactivation efficiency was confirmed, including resistant pathogens such as human rhinovirus and Clostridium difficile. In addition, we tested other filter attributes, such as filtration efficiency and breathability, to determine the optimal layer for salt coating and its effects on performance. Our findings revealed that the outer layer of a conventional face mask plays a crucial role in contact transmission through contaminated face masks and respirators. Through contact transmission experiments using droplets involving three types of contaminants (fluorescent dyes, bacteria, and viruses), the salt-coated stacked SB fabric demonstrated a superior effect in preventing contact transmission compared to SB or meltblown polypropylene fabrics─an issue challenging to existing masks. Our results demonstrate that the use of salt-coated stacked SB fabrics as (i) the outer layer of a mask and (ii) a mask cover over a mask enhances overall filter performance against infectious droplets, achieving high pathogen inactivation and low contact-based transmission while maintaining breathability.

Non-Invasive Vaccines: Challenges in Formulation and Vaccine Adjuvants.

Given the limitations of conventional invasive vaccines, such as the requirement for a cold chain system and trained personnel, needle-based injuries, and limited immunogenicity, non-invasive vaccines have gained significant attention. Although numerous approaches for formulating and administrating non-invasive vaccines have emerged, each of them faces its own challenges associated with vaccine bioavailability, toxicity, and other issues. To overcome such limitations, researchers have created novel supplementary materials and delivery systems. The goal of this review article is to provide vaccine formulation researchers with the most up-to-date information on vaccine formulation and the immunological mechanisms available, to identify the technical challenges associated with the commercialization of non-invasive vaccines, and to guide future research and development efforts.

Highly Effective Salt-Activated Alcohol-Based Disinfectants with Enhanced Antimicrobial Activity.

Surfaces contaminated with pathogens raise concerns about the increased risk of disease transmission and infection. To clean biocontaminated surfaces, alcohol-based disinfectants have been predominantly used for disinfecting high-touch areas in diverse settings. However, due to its limited antimicrobial activities and concern over the emergence of alcohol-tolerant strains, much effort has been made to develop highly efficient disinfectant formulations. In this study, we hypothesize that the addition of a physical pathogen inactivation mechanism by salt recrystallization (besides the existing chemical inactivation mechanism by alcohol in such formulations) can improve inactivation efficiency by preventing the emergence of alcohol tolerance. To this end, we employed the drying-induced salt recrystallization process to implement the concept of highly efficient alcohol-based disinfectant formulations. To identify the individual and combined effects of isopropyl alcohol (IPA) and NaCl, time-dependent morphological/structural changes of various IPA solutions containing NaCl have been characterized by optical microscopy/X-ray diffraction analysis. Their antimicrobial activities have been tested on surfaces (glass slide, polystyrene Petri dish, and stainless steel) contaminated with Gram-positive/negative bacteria (methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica subsp. enterica Typhimurium) and viruses (A/PR8/34 H1N1 influenza virus and HCoV-OC43 human coronavirus). We found that additional salt crystallization during the drying of the alcohol solution facilitated stronger biocidal effects than IPA-only formulations, regardless of the types of solid surfaces and pathogens, including alcohol-tolerant strains adapted from wild-type Escherichia coli MG1655. Our findings can be useful in developing highly effective disinfectant formulations by minimizing the use of toxic antimicrobial substances to improve public health and safety.

Photopolymerizable, Universal Antimicrobial Coating to Produce High-Performing, Multifunctional Face Masks.

COVID-19 poses a major threat to global health and socioeconomic structures, and the need for a highly effective, antimicrobial face mask has been considered a major challenge for protection against respiratory diseases. Here, we report the development of a universal, antiviral, and antibacterial material that can be dip-/spray-coated over conventional mask fabrics to exhibit antimicrobial activities. Our data shows that antimicrobial fabrics rapidly inactivated multiple types of viruses, i.e., human (alpha/beta) coronaviruses, the influenza virus, and bacteria, irrespective of their modes of transmission (aerosol or droplet). This research provides an immediate method to contain infectious diseases, such as COVID-19.

Study of the Pathogen Inactivation Mechanism in Salt-Coated Filters.

As COVID-19 exemplifies, respiratory diseases transmitted through aerosols or droplets are global threats to public health, and respiratory protection measures are essential first lines of infection prevention and control. However, common face masks are single use and can cause cross-infection due to the accumulated infectious pathogens. We developed salt-based formulations to coat membrane fibers to fabricate antimicrobial filters. Here, we report a mechanistic study on salt-induced pathogen inactivation. The salt recrystallization following aerosol exposure was characterized over time on sodium chloride (NaCl), potassium sulfate (K2SO4), and potassium chloride (KCl) powders and coatings, which revealed that NaCl and KCl start to recrystallize within 5 min and K2SO4 within 15 min. The inactivation kinetics observed for the H1N1 influenza virus and Klebsiella pneumoniae matched the salt recrystallization well, which was identified as the main destabilizing mechanism. Additionally, the salt-coated filters were prepared with different methods (with and without a vacuum process), which led to salt coatings with different morphologies for diverse applications. Finally, the salt-coated filters caused a loss of pathogen viability independent of transmission mode (aerosols or droplets), against both DI water and artificial saliva suspensions. Overall, these findings increase our understanding of the salt-recrystallization-based technology to develop highly versatile antimicrobial filters.

Salt coatings functionalize inert membranes into high-performing filters against infectious respiratory diseases.

Respiratory protection is key in infection prevention of airborne diseases, as highlighted by the COVID-19 pandemic for instance. Conventional technologies have several drawbacks (i.e., cross-infection risk, filtration efficiency improvements limited by difficulty in breathing, and no safe reusability), which have yet to be addressed in a single device. Here, we report the development of a filter overcoming the major technical challenges of respiratory protective devices. Large-pore membranes, offering high breathability but low bacteria capture, were functionalized to have a uniform salt layer on the fibers. The salt-functionalized membranes achieved high filtration efficiency as opposed to the bare membrane, with differences of up to 48%, while maintaining high breathability (> 60% increase compared to commercial surgical masks even for the thickest salt filters tested). The salt-functionalized filters quickly killed Gram-positive and Gram-negative bacteria aerosols in vitro, with CFU reductions observed as early as within 5 min, and in vivo by causing structural damage due to salt recrystallization. The salt coatings retained the pathogen inactivation capability at harsh environmental conditions (37 °C and a relative humidity of 70%, 80% and 90%). Combination of these properties in one filter will lead to the production of an effective device, comprehensibly mitigating infection transmission globally.

Halloysite nanotube-embedded microparticles for intestine-targeted co-delivery of biopharmaceuticals.

Microparticles (MPs) with pH-responding macropores have recently proved their significance for the delivery of vulnerable biomolecules for oral drug administration. The previous MP systems were proven to provide enhanced protection against the gastric environment, however, their application is hindered due to insufficient loading efficiencies and deficient penetration capabilities of encapsulated drugs across the mucus barrier. Here, we report a new co-delivery approach based on amine-functionalized halloysite nanotube (HNT)-embedded MPs (amine-HNT-MPs) with pH-responding macropores specifically designed to deal with the mucus barrier at the absorption site. The mean diameter and polydispersity index of the pored MPs were measured by a particle size analyzer to be 37.6 ± 1.3 µm and 1.15, respectively. The drug loading capacity of the co-delivery system was shown to be 50-times higher than previously reported pored MPs. Fluorescence microscopy analysis of sulforhodamine B (into a hollow interior of HNTs)/ fluorescent nanoparticles (into a hollow interior of MPs)-encapsulated MPs confirmed biphasic release behavior due to pH-dependent pore closing/opening in the simulated gastrointestinal (GI) digestive conditions. To verify the protective effect of the co-delivery system, bromelain and lactase were loaded into HNTs and MPs, respectively, and found to exhibit 94.5 ± 3.3% (bromelain) and 70 ± 14.1% (lactase) functional activity in simulated GI tract conditions. The considerable improvement in the stability of the encapsulated enzymes against gastric conditions are attributed to the efficient pore sealing of the co-delivery system after the encapsulation of enzymes and maintenance of these closed pores in the gastric environment. Furthermore, the mucolytic enzyme (i.e. bromelain)-encapsulated co-delivery system was found to enhance mucopenetration of the encapsulated drug from histological analysis using ex vivo porcine intestine tissue. Therefore, the new microencapsulation design proposed in this study provides a promising solution to the major issues hampering the wide-spread application of MPs in the development of oral drug formulations for biopharmaceuticals and vaccines.

Protection induced by malaria virus-like particles containing codon-optimized AMA-1 of Plasmodium berghei.

BACKGROUND: Despite the extensive endeavours, developing an effective malaria vaccine remains as a great challenge. Apical membrane antigen 1 (AMA-1) located on the merozoite surface of parasites belonging to the genus Plasmodium is involved in red blood cell invasion. METHODS: Influenza virus-like particle (VLP) vaccines containing codon-optimized or native (non-codon optimized) AMA-1 from Plasmodium berghei were generated. VLP-induced protective immunity was evaluated in a mouse model. RESULTS: Mice immunized with VLP vaccine containing the codon-optimized AMA-1 elicited higher levels of P. berghei-specific IgG and IgG2a antibody responses compared to VLPs containing non-codon optimized AMA-1 before and after challenge infection. Codon-optimized AMA-1 VLP vaccination induced higher levels of CD4+ T cells, CD8+ T cells, B cells, and germinal centre cell responses compared to non-codon optimized AMA-1 VLPs. Importantly, the codon-optimized AMA-1 VLP vaccination showed lower body weight loss, longer survival and a significant decrease in parasitaemia compared to non-codon optimized VLP vaccination. CONCLUSION: Overall, VLP vaccine expressing codon-optimized AMA-1 induced better protective efficacy than VLPs expressing the non-codon optimized AMA-1. Current findings highlight the importance of codon-optimization for vaccine use and its potential involvement in future malaria vaccine design strategies.

Influenza Virus-Like Particles Presenting both Toxoplasma gondii ROP4 and ROP13 Enhance Protection against T. gondii Infection.

Rhoptry organelle proteins (ROPs) secreted by Toxoplasma gondii (T. gondii) play a critical role during parasite invasion into host cells. In this study, virus-like particles (VLPs) vaccines containing ROP4 and/or ROP13 together with influenza M1 were generated. ROP4+ROP13 VLPs were produced by combining ROP4 VLPs with ROP13 VLPs, and ROP(4 + 13) VLPs by co-infecting insect cells with recombinant baculovirus expressing ROP4 or ROP13. Mice intranasally immunized with ROP(4 + 13) VLPs showed significantly higher levels of IgG, IgG1, IgG2a and IgA antibody responses in sera compared to ROP4+ROP13VLPs. Upon challenge infection by oral route, mice immunized with ROP(4 + 13) VLPs elicited higher levels of IgG and IgA antibody responses in fecal, urine, intestine and vaginal samples as well as CD4+ T, CD8+ T cells, and germinal center B cell responses compared to other type of vaccines, ROP4 VLPs, ROP13 VLPs, and ROP4+ROP13 VLPs. ROP(4 + 13) VLPs vaccination showed a significant decrease in the size and number of cyst in the brain and less body weight loss compared to combination ROP4+ROP13 VLPs upon challenge infection with T. gondii ME49. These results indicated that the ROP(4 + 13) VLPs vaccination provided enhanced protection against T. gondii infection compared to ROP4+ROP13 VLPs, providing an important insight into vaccine design strategy for T. gondii VLPs vaccines.

Co-Delivery of M2e Virus-Like Particles with Influenza Split Vaccine to the Skin Using Microneedles Enhances the Efficacy of Cross Protection.

It is a high priority to develop a simple and effective delivery method for a cross-protective influenza vaccine. We investigated skin immunization by microneedle (MN) patch with human influenza split vaccine and virus-like particles containing heterologous M2 extracellular (M2e) domains (M2e5x virus-like particles (VLP)) as a cross-protective influenza vaccine candidate. Co-delivery of influenza split vaccine and M2e5x VLP to the skin by MN patch was found to confer effective protection against heterosubtypic influenza virus by preventing weight loss and reducing lung viral loads. Compared to intramuscular immunization, MN-based delivery of combined split vaccine and M2e5x VLPs shaped cellular immune responses toward T helper type 1 responses increasing IgG2a isotype antibodies as well as IFN-γ producing cells in mucosal and systemic sites. This study provides evidence that potential immunological and logistic benefits of M2e5x VLP with human influenza split vaccine delivered by MN patch can be used to develop an easy-to-administer cross-protective influenza vaccine.

Challenges and Recent Progress in Oral Drug Delivery Systems for Biopharmaceuticals.

Routes of drug administration and the corresponding physicochemical characteristics of a given route play significant roles in therapeutic efficacy and short term/long term biological effects. Each delivery method has favorable aspects and limitations, each requiring a specific delivery vehicles design. Among various routes, oral delivery has been recognized as the most attractive method, mainly due to its potential for solid formulations with long shelf life, sustained delivery, ease of administration and intensified immune response. At the same time, a few challenges exist in oral delivery, which have been the main research focus in the field in the past few years. The present work concisely reviews different administration routes as well as the advantages and disadvantages of each method, highlighting why oral delivery is currently the most promising approach. Subsequently, the present work discusses the main obstacles for oral systems and explains the most recent solutions proposed to deal with each issue.

Virus-Like Particles Are a Superior Platform for Presenting M2e Epitopes to Prime Humoral and Cellular Immunity against Influenza Virus.

Influenza virus M2 protein has a highly conserved ectodomain (M2e) as a cross-protective antigenic target. We investigated the antigenic and immunogenic properties of tandem repeat M2e (5xM2e) proteins and virus-like particles (5xM2e VLP) to better understand how VLP and protein platform vaccines induce innate and protective adaptive immune responses. Despite the high antigenic properties of 5xM2e proteins, the 5xM2e VLP was superior to 5xM2e proteins in inducing IgG2a isotype antibodies, T cell responses, plasma cells and germinal center B cells as well as in conferring cross protection. Mice primed with 5xM2e VLP were found to be highly responsive to 5xM2e protein boost, overcoming the low immunogenicity and protective efficacy of 5xM2e proteins. Immunogenic differences between VLPs and proteins in priming immune responses might be due to an intrinsic ability of 5xM2e VLP to stimulate dendritic cells secreting T helper type 1 (Th1) cytokines. We also found that 5xM2e VLP was effective in inducing inflammatory cytokines and chemokines, and in recruiting macrophages, monocytes, neutrophils, and CD11b⁺ dendritic cells at the injection site. Therefore, this study provides evidence that 5xM2e VLP is an effective vaccine platform, inducing cross-protection by stimulating innate and adaptive immune responses.

Macropored microparticles with a core-shell architecture for oral delivery of biopharmaceuticals.

Microparticles (MPs) have been extensively researched as a potential drug delivery vehicle. Here, we investigated the fabrication of MPs with pH-responsive macropores and evaluated their potential applicability in developing solid oral drug formulations. Our previous study showed that macropored MPs, made of Eudragit® L100-55, could encapsulate 100 nm, 1 µm, and 4 µm sized fluorescent beads-model drugs that are mimicking vaccines, bacteria, and cells. In the present study, closed-pored MPs after freeze-drying were coated with a gastric soluble Eudragit® EPO layer to protect MPs in the simulated pregastric environment. Subsequently, drug encapsulated MPs maintained their intact closed-pored structure in the simulated gastric environment and exhibited a rapid release in the simulated intestine environment. Our MP system was found to provide a significantly higher level of protection to the encapsulated lactase enzyme compared to the control sample (i.e. without using MPs). Real-time fluorescence microscopy analysis showed that macropored MPs released encapsulated drugs in a burst-release pattern and in a size-independent manner. This work shows that our proposed EPO-coated MPs with pH-responsive macropores can meet the challenges posed by the multiple physiological environments of the digestive tract and be used in developing highly effective solid oral drug/vaccine formulations.

Facile fabrication of microparticles with pH-responsive macropores for small intestine targeted drug formulation.

Oral drugs present the most convenient, economical, and painless route for self-administration. Despite commercialization of multiple technologies relying on micro- and nanocrystalline drugs, research on microparticles (MPs) based oral biopharmaceuticals delivery systems has still not culminated well enough in commercial products. This is largely due to the drugs being exposed to the destabilizing environment during MP synthesis process, and partly because of complicated process conditions. Hence, we developed a solvent swelling-evaporation method of producing pH-responsive MPs with micron-sized macropores using poly(methacrylic acid-co-ethyl acrylate) in 1:1 ratio (commercial name: Eudragit® L100-55 polymer). We investigated the effects of temperature and evaporation time on pore formation, freeze-drying induced pore closure, and the release profile of model drugs (fluorescent beads, lactase, and pravastatin sodium) encapsulated MPs in simulated gastrointestinal tract conditions. Encapsulated lactase/pravastatin maintained >60% of their activity due to the preservation of pore closure, which proved the potential of this proof-of-concept microencapsulation system. Importantly, the presence of macropores on MPs can be beneficial for easy drug loading, and solve the problem of bioactivity loss during the conventional MP fabrication-drug encapsulation steps. Therefore, pH-sensing MPs with macropores can contribute to the development of oral drug formulations for a wide variety of drugs and bio-macromolecules, having a various size ranging from genes to micron-sized ingredients with high therapeutic efficacy.

Antioxidant-based synergistic eradication of methicillin-resistant Staphylococcus aureus (MRSA) biofilms with bacitracin.

Biofilms of methicillin-resistant Staphylococcus aureus (MRSA) have serious clinical implications. However, it is difficult to eradicate MRSA biofilms due to the increased tolerance to antimicrobials of biofilms. In this study, we investigated the synergistic anti-biofilm effect of the combination of octyl gallate (OG), an antioxidant approved by the US Food and Drug Administration (FDA) as a food additive, and bacitracin, an antimicrobial peptide commonly used in topical antimicrobial ointments. The results of biofilm assays showed that OG enabled bacitracin at concentrations as low as 10-3 U/ml to inhibit biofilm formation in MRSA. A confocal microscopic analysis exhibited that the combination of bacitracin and OG suppressed biofilm formation in MRSA highly effectively compared to the single treatment of either bacitracin or OG. The synergistic anti-biofilm activity of bacitracin and OG was also confirmed in MRSA strains from humans, including USA300, which is the predominant clone of community-associated MRSA in the US. To the best of our knowledge, this is the first report about the synergistic anti-biofilm activity of an antimicrobial peptide and an antioxidant against MRSA.

Protection induced by virus-like particle vaccine containing tandem repeat gene of respiratory syncytial virus G protein.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, young children and the elderly. However, there is no licensed vaccine available against RSV infection. In this study, we generated virus-like particle (VLP) vaccine and investigated the vaccine efficacy in a mouse model. For VLP vaccines, tandem gene (1-780 bp) for V1 VLPs and tandem repeat gene (repeated 450-780 bp) for V5 VLPs were constructed in pFastBacTM vectors, respectively. Influenza matrix protein 1 (M1) was used as a core protein in the VLPs. Notably, upon challenge infection, significantly lower virus loads were measured in the lung of mice immunized with V1 or V5 VLPs compared to those of naïve mice and formalin-inactivated RSV immunized control mice. In particular, V5 VLPs immunization showed significantly lower virus titers than V1 VLPs immunization. Furthermore, V5 VLPs immunization elicited increased memory B cells responses in the spleen. These results indicated that V5 VLP vaccine containing tandem repeat gene protein provided better protection than V1 VLPs with significantly decreased inflammation in the lungs. Thus, V5 VLPs could be a potential vaccine candidate against RSV.

Immunogenicity and efficacy of replication-competent recombinant influenza virus carrying multimeric M2 extracellular domains in a chimeric hemagglutinin conjugate.

Current influenza vaccines provide hemagglutinin (HA) strain-specific protection. To improve cross protection, we engineered replication-competent influenza A virus to express tandem repeats of heterologous M2 extracellular (M2e) domains in a chimeric HA. M2e epitopes conjugated to HA glycoproteins (M2e4x-HA) were found to be expressed on the surfaces of a replicable influenza virus as examined by electron microscopy. The recombinant influenza virus containing M2e4x-HA was moderately attenuated but superior to the parental virus in inducing M2e specific antibodies without compromising HA immunogenicity. Recombinant influenza virus immune mice showed better cross protection than parental virus immune mice. Immune sera from the mice with inoculation of live recombinant influenza virus expressing M2e4x-HA were effective in conferring protection against H1, H3, and H5 subtype influenza viruses. This study indicates that recombinant influenza virus expressing conserved protective epitopes in an HA chimeric form can provide a new approach for improving the efficacy of influenza vaccines.

Respiratory Protection against Pandemic and Epidemic Diseases.

Respiratory protection against airborne pathogens is crucial for pandemic/epidemic preparedness in the context of personal protection, healthcare systems, and governance. We expect that the development of technologies that overcome the existing challenges in current respiratory protective devices will lead to a timely and effective response to the next outbreak.

Smart Microparticles with a pH-responsive Macropore for Targeted Oral Drug Delivery.

The development of a smart microencapsulation system programmed to actively respond to environmental pH change has long been recognized a key technology in pharmaceutical and food sciences. To this end, we developed hollow microparticles (MPs) with self-controlled macropores that respond to environmental pH change, using an Oil-in-Water emulsion technique, for oral drug delivery. We observed that freeze-drying of MPs induced closure of macropores. The closing/opening behavior of macropores was confirmed by exposing MPs encapsulating different ingredients (sulforhodamine b, fluorescent nanoparticles, and lactase) to simulated gastrointestinal (GI) fluids. MPs maintained their intact, closed pore structure in gastric pH, and subsequent exposure to intestinal pH resulted in pore opening and ingredients release. Further, MPs displayed higher protection (>15 times) than commercial lactase formulation, indicating the protective ability of the system against harsh GI conditions. This study showed development of a hybrid MP system combining the advantages of solid particles and hollow capsules, exhibiting easy solvent-free loading mechanism and smart protection/release of encapsulates through controllable macropores. Ultimately, our MPs system strives to usher a new research area in smart drug delivery systems and advance the current oral drug delivery technology by solving major challenges in targeted delivery of pH-sensitive therapeutics.

Protection induced by virus-like particles containing Toxoplasma gondii microneme protein 8 against highly virulent RH strain of Toxoplasma gondii infection.

Toxoplasma gondii (T. gondii) microneme protein 8 (MIC8) represents a novel, functional distinct invasion factor. In this study, we generated virus-like particles (VLPs) targeting Toxoplasma gondii MIC8 for the first time, and investigated the protection against highly virulent RH strain of T. gondii in a mouse model. We found that VLP vaccination induced Toxoplasma gondii-specific IgG and IgG1 antibody responses in the sera. Upon challenge infection with RH strain of T. gondii tachyzoites, vaccinated mice showed a significant increase of both IgG antibodies in sera and IgA antibodies in feces compared to those before challenge, and a rapid expansion of both germinal center B cell (B220+, GL7+) and T cell (CD4+, CD8+) populations. Importantly, intranasally immunized mice showed higher neutralizing antibodies and displayed no proinflammatory cytokine IFN-γ in the spleen. Mice were completely protected from a lethal challenge infection with the highly virulent T. gondii (RH) showing no body weight loss (100% survival). Our study shows the effective protection against T. gondii infection provided by VLPs containing microneme protein 8 of T. gondii, thus indicating a potential T. gondii vaccine candidate.